Organic compounds

ABSTRACT

A method of inhibiting dipeptidyl peptidase-IV comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound of formulae (I A), (I B), (X A), (X B), (Y A) or (Y or B), 
     wherein R′ represents 
                         
and R″ represents hydrogen, hydroxy, C 1 -C 7 alkoxy, C 1 -C 8 -alkanoyloxy, or R 5 R 4 N—CO—O—, where R 4  and R 5  independently are C 1 -C 7 alkyl or phenyl which is unsubstituted or substituted by a substitutent selected from C 1 -C 7 alkyl, C 1 -C 7 alkoxy, halogen and trifluoromethyl and where R 4  additionally is hydrogen; or R 4  and R 5  together represent C 3 -C 6 alkylene,
 
in free form or in form of a pharmaceutically acceptable acid addition salt.

This application is a continuation application of Ser. No. 12/744,510,filed May 25, 2010, which is a National Phase application ofPCT/EP/2008/066159, filed Nov. 25, 2008, which claims benefit ofProvisional Application No. 61/004,846, filed Nov. 30, 2007, which intheir entirety are herein incorporated by reference.

The present invention provides new dipeptidyl peptidase-IV (DPP-IV)inhibitors which are effective in treating conditions mediated byDPP-IV. It was discovered that DPP-IV is responsible for inactivatingglucagon-like peptide-1 (GLP-1). Since GLP-1 is a major stimulator ofpancreatic insulin secretion and has direct beneficial effects onglucose disposal, DPP-IV inhibition appears to represent an attractiveapproach for treating conditions such as non-insulin-dependent diabetesmellitus (NIDDM).

The instant invention relates to a compound of formulae (I A), (I B), (XA), (X B), (Y A) or (Y B) (compounds of the invention)

wherein R′ represents

-   -   and R″ represents hydrogen, hydroxy, C₁-C₇alkoxy,        C₁-C₈-alkanoyloxy, or R₅R₄N—CO—O—, where R₄ and R₅ independently        are C₁-C₇alkyl or phenyl which is unsubstituted or substituted        by a substitutent selected from C₁-C₇alkyl, C₁-C₇alkoxy, halogen        and trifluoromethyl and where R₄ additionally is hydrogen; or R₄        and R₅ together represent C₃-C₆alkylene;    -   in free form or in form of a pharmaceutically acceptable acid        addition salt.

The invention also concerns a compound of formulae (I A), (I B), (X A),(X B), (Y A) or (Y B) as hereinabove described, wherein R″ representshydrogen.

In preferred embodiment, the compounds of formulae (I A), (I B), (X A),(X B), (Y A) or (Y B) as hereinabove described are in a substantiallypure form.

The compounds of the invention can exist in free form or in acidaddition salt form. Pharmaceutically acceptable (i.e., non-toxic,physiologically acceptable) salts are preferred, although other saltsare also useful, e.g., in isolating or purifying the compounds of thisinvention. Although the preferred acid addition salts are thehydrochlorides, salts of methanesulfonic, sulfuric, phosphoric, citric,lactic and acetic acid may also be utilized.

The compounds of the invention may exist in the form of optically activeisomers or diastereoisomers and can be separated and recovered byconventional techniques, such as chromatography.

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

The term “alkyl” refers to straight or branched chain hydrocarbon groupshaving 1 to 10 carbon atoms, preferably 1 to 7 carbon atoms, mostpreferably 1 to 5 carbon atoms. Exemplary alkyl groups include methyl,ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl andthe like.

The term “alkanoyl” refers to alkyl-C(O)—.

The term “substituted adamantyl” refers to adamantyl, i.e. 1- or2-adamantyl, substituted by one or more, for example two, substitutentsselected from alkyl, —OR₁ or —NR₂R₃; where R₂ and R₃ are independentlyhydrogen, alkyl, (C₁-C₈-alkanoyl), carbamyl, or —CO—NR₄R₅; where R₄ andR₅ are independently alkyl, unsubstituted or substituted aryl and whereone of R₄ and R₅ additionally is hydrogen or R₄ and R₅ togetherrepresent C₂-C₇alkylene.

The term “aryl” preferably represents phenyl. Substituted phenylpreferably is phenyl substituted by one or more, e.g. two, substitutentsselected from e.g. alkyl, alkoxy, halogen and trifluoromethyl.

The term “alkoxy” refers to alkyl-O—.

The term “halogen” or “halo” refers to fluorine, chlorine, bromine andiodine.

The term “alkylene” refers to a straight chain bridge of 2 to 7 carbonatoms, preferably of 3 to 6 carbon atoms, most preferably 5 carbonatoms.

The term “substantially pure” is understood in the context of thepresent invention to mean substantially free of biological material suchas found in the blood, especially less than 10%, preferably less than1%, and most preferably free of such biological material.

The compounds of the invention may be prepared e.g. by a process whichcomprises coupling a reactive (2-cyanopyrrolidino)carbonylmethylenecompound with an appropriate substituted amine; more particularly, forthe preparation of the compounds of formula I, it comprises reacting acompound of formula II

wherein Y is a reactive group (preferably a halogen such as bromine,chlorine or iodine) with a compound of formula IIINH₂(CH₂)_(n)—R  IIIwherein R is as defined above, and recovering the resultant compound offormulae (I A), (I B), (X A), (X B), (Y A) or (Y B), in free form or inacid addition salt form.

Alternative processes to prepare precursors of the compounds of formulaYA or YB are described in the patent application WO 01/068603, or WO05/095339 for the compounds of formula XA or XB. The below describedprocess can be used to obtain the O-glucuronide form of the compoundsdescribed in the patent application WO 01/068603, or WO 05/095339 andwherein R′ is —OH.

The R′ moiety can be attached to the chemical structure by any of themethod well known by the person skilled in the art or by the processdescribed herein after for the structures of the chemical formula IA orIB. Via preparative bioconversion using rat liver homogenate as thecatalyst, the O-glucuronide of vildagliptin have been prepared asdescribed herein after.

Vildagliptin (NVP-LAF237-NX or Galvus®) is a new oral hypoglycemic(anti-diabetic) drug of the dipeptidyl peptidase-4 (DPP-4) inhibitorclass currently undergoing regulatory review by the U.S. FDA. Theapplicant has surprisingly discovered that the O-glucuronide ofvildagliptin (Figure AA) remains as active as vildagliptin. Theapplicant has surprisingly discovered that the O-glucuronide ofvildagliptin in addition to be equally potent to vildagliptin atinhibiting the DPP-4 enzyme, is less potent at inhibiting DPP-8 or DPP-9enzymes. Its particularly unexpected that substituting the adamantlygroup of a DPP-4 inhibitors would provide improved pharmacologicalprofile. The O-glucuronide of vildagliptin can provide furtherpharmacokinetic or pharmacological advantages e.g. less side effects,better bioavailability.

In a most preferred embodiment, the O-glucuronide of vildagliptin(Figure AA) is in a substantially pure form.

The process of the invention may be effected in conventional manner. Forexample, the compound of formula II is reacted with 1 to 3 equivalents,preferably 3 equivalents of a primary amine of formula III. The reactionis conveniently conducted in the presence of an inert, organic solvent,such as methylene chloride or a cyclic ether such as tetrahydrofuran.The temperature preferably is of from about 0° to about 35° C.,preferably between about 0° and about 25° C.

The compounds of the invention may be isolated from the reaction mixtureand purified in conventional manner, e.g. by chromatography.

The starting materials may also be prepared in conventional manner. Thecompounds of formula II may be prepared by the following two-stepreaction scheme:

Step 1 involves the reaction of the pyrrolidine of formula IV with aslight molar excess of a haloacetylhalide such as bromoacetylbromide orchloroacetylchloride and a base such as potassium carbonate ortriethylamine. The reaction conveniently is conducted in the presence ofan inert, organic solvent, such as tetrahydrofuran or a chlorinated,aliphatic hydrocarbon such as methylene chloride, at a temperature offrom about 0° to about 25° C., preferably at a temperature between about0° and about 15° C.

Step 2 concerns the dehydration of the compound of formula V, preparedin Step 1, with 1 to 2 equivalents of trifluoroacetic anhydride (TFAA).The dehydration preferably is conducted in the presence of an inert,organic solvent such as tetrahydrofuran or a chlorinated, aliphatichydrocarbon such as methylene chloride, at a temperature of from about0° to about 25° C., preferably at a temperature between about 0° andabout 15° C.

Insofar as its preparation is not particularly described herein, acompound used as starting material is known or may be prepared fromknown compounds in known manner or analogously to known methods oranalogously to methods described in the Example.

For example, the primary amine compounds of formula III are known andmay be prepared by procedures documented in the literature, for example,Khim.-Farm. Zh. (1986), 20(7), 810-15.

Finally, compounds of the invention are either obtained in the freeform, or as a salt thereof if salt forming groups are present.

Compounds of the invention having basic groups can be converted intoacid addition salts, especially pharmaceutically acceptable acidaddition salts. These are formed, for example, with inorganic acids,such as mineral acids, for example sulfuric acid, a phosphoric orhydrohalic acid, or with organic carboxylic acids. Preferred are saltsformed with hydrochloric acid.

In view of the close relationship between the free compounds and thecompounds in the form of their salts, whenever a compound is referred toin this context, a corresponding salt is also intended, provided such ispossible or appropriate under the circumstances.

The compounds, including their salts, can also be obtained in the formof their hydrates, or include other solvents used for theircrystallization.

The instant invention also includes pharmaceutical compositions, forexample, useful in inhibiting DPP-IV, comprising a pharmaceuticallyacceptable carrier or diluent and a therapeutically effective amount ofa compound of formula I, or a pharmaceutically acceptable acid additionsalt thereof.

In still another embodiment, the instant invention provides a method ofinhibiting DPP-IV comprising administering to a mammal in need of suchtreatment a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable acid addition salt thereof.

In a further embodiment, the instant invention provides a method oftreating conditions mediated by DPP-IV inhibition comprisingadministering to a mammal in need of such treatment a therapeuticallyeffective amount of a compound of the invention above, or apharmaceutically acceptable acid addition salt thereof.

The present invention also relates to the use of a compound according tothe instant invention or a pharmaceutically acceptable salt thereof e.g.for the manufacture of a medicament for the prevention or treatment ofdiseases or conditions associated with elevated levels of DPP-IV.

As indicated above, all of the compounds of formulae (I A), (I B), (XA), (X B), (Y A) or (Y B), and their corresponding pharmaceuticallyacceptable acid addition salts, are useful in inhibiting DPP-IV. Theability of the compounds of the invention, and their correspondingpharmaceutically acceptable acid addition salts, to inhibit DPP-IV maybe demonstrated employing the Caco-2 DPP-IV Assay which measures theability of test compounds to inhibit DPP-IV activity from human coloniccarcinoma cell extracts. The human colonic carcinoma cell line Caco-2was obtained from the American Type Culture Collection (ATCC HTB 37).Differentiation of the cells to induce DPP-IV expression wasaccomplished as described by Reisher, et al. in an article entitled“Increased expression of intestinal cell line Caco-2” in Proc. Natl.Acad. Sci., Vol. 90, pgs. 5757-5761 (1993). Cell extract is preparedfrom cells solubilized in 10 mM Tris HCl, 0.15 M NaCl, 0.04 t.i.u.aprotinin, 0.5% nonidet-P40, pH 8.0, which is centrifuged at 35,000 gfor 30 min. at 4° C. to remove cell debris. The assay is conducted byadding 20 μg solubilized Caco-2 protein, diluted to a final volume of125 μl in assay buffer (25 mM Tris HCl pH 7.4, 140 mM NaCl, 10 mM KCl,1% bovine serum albumin) to microtiter plate wells. After a 60 min.incubation at room temperature, the reaction is initiated by adding 25μl of 1 mM substrate (H-Alanine-Proline-pNA; pNA is p-nitroaniline). Thereaction is carried out at room temperature for 10 minutes after whichtime a 19 μl volume of 25% glacial acetic acid is added to stop thereaction. Test compounds are typically added as 30 μl additions and theassay buffer volume is reduced to 95 μl. A standard curve of freep-nitroaniline is generated using 0-500 μM solutions of free pNA inassay buffer. The curve generated is linear and is used forinterpolation of substrate consumption (catalytic activity in nmolessubstrate cleaved/min). The endpoint is determined by measuringabsorbance at 405 nm in a Molecular Devices UV Max microtiter platereader.

The potency of the test compounds as DPP-IV inhibitors, expressed asIC₅₀, is calculated from 8-point, dose-response curves using a4-parameter logistic function.

The ability of the compounds of the invention, and their correspondingpharmaceutically acceptable acid addition salts, to inhibit DPP-IV mayalso be demonstrated by measuring the effects of test compounds onDPP-IV activity in human and rat plasma employing a modified version ofthe assay described by Kubota, et al. in an article entitled“Involvement of dipeptidylpeptidase IV in an in vivo immune response” inClin. Exp. Immunol., Vol. 89, pgs. 192-197 (1992). Briefly, 5 μl ofplasma are added to 96-well flat-bottom microtiter plates (Falcon),followed by the addition of 5 μl of 80 mM MgCl₂ in incubation buffer (25mMHEPES, 140 mM NaCl, 1% RIA-grade BSA, pH 7.8). After a 60 min.incubation at room temperature, the reaction is initiated by theaddition of 10 μl of incubation buffer containing 0.1 mM substrate(H-Glycine-Proline-AMC; AMC is 7-amino-4-methylcoumarin). The plates arecovered with aluminum foil (or kept in the dark) and incubated at roomtemperature for 20 min. After the 20 min. reaction, fluorescence ismeasured using a CytoFluor 2350 fluorimeter (Excitation 380 nm Emission460 nm; sensitivity setting 4). Test compounds are typically added as 2μl additions and the assay buffer volume is reduced to 13 μl. Afluorescence-concentration curve of free AMC is generated using 0-50 μMsolutions of AMC in assay buffer. The curve generated is linear and isused for interpolation of substrate consumption (catalytic activity innmoles substrate cleaved/min). As with the previous assay, the potencyof the test compounds as DPP-IV inhibitors, expressed as IC₅₀, iscalculated from 8-point, dose-response curves using a 4 parameterlogistic function.

In view of their ability to inhibit DPP-IV, the compounds of theinvention, and their corresponding pharmaceutically acceptable acidaddition salts, are useful in treating conditions mediated by DPP-IVinhibition. Based on the above and findings in the literature, it isexpected that the compounds disclosed herein are useful in the treatmentof conditions such as non-insulin-dependent diabetes mellitus,arthritis, obesity, allograft transplantation andcalcitonin-osteoporosis. In addition, based on the roles ofglucagon-like peptides (such as GLP-1 and GLP-2) and their associationwith DPP-IV inhibition, it is expected that the compounds disclosedherein are useful for example, to produce a sedative or anxiolyticeffect, or to attenuate post-surgical catabolic changes and hormonalresponses to stress, or to reduce mortality and morbidity aftermyocardial infarction, or in the treatment of conditions related to theabove effects which may be mediated by GLP-1 and/or GLP-2 levels.

More specifically, for example, the compounds of the invention, andtheir corresponding pharmaceutically acceptable acid addition salts,improve early insulin response to an oral glucose challenge and,therefore, are useful in treating non-insulin-dependent diabetesmellitus. The ability of the compounds of the invention, and theircorresponding pharmaceutically acceptable acid addition salts, toimprove early insulin response to an oral glucose challenge may bemeasured in insulin resistant rats according to the following method:

Male Sprague-Dawley rats that had been fed a high fat diet (saturatedfat=57% calories) for 2-3 weeks were fasted for approximately 2 hours onthe day of testing, divided into groups of 8-10, and dosed orally with10 μmol/kg of the test compounds in CMC. An oral glucose bolus of 1 g/kgwas administered 30 minutes after the test compound directly into thestomach of the test animals. Blood samples, obtained at varioustimepoints from chronic jugular vein catheters, were analyzed for plasmaglucose and immunoreactive insulin (IRI) concentrations, and plasmaDPP-IV activity. Plasma insulin levels were assayed by a double antibodyradioimmunoassay (RIA) method using a specific anti-rat insulin antibodyfrom Linco Research (St. Louis, Mo.). The RIA has a lower limit ofdetection of 0.5 μU/mL with intra- and inter-assay variations of lessthan 5%. Data are expressed as % increase of the mean of the controlanimals. Upon oral administration, each of the compounds testedamplified the early insulin response which led to an improvement inglucose tolerance in the insulin resistant test animals. The followingresults were obtained:

The precise dosage of the compounds of the invention, and theircorresponding pharmaceutically acceptable acid addition salts, to beemployed for treating conditions mediated by DPP-IV inhibition dependsupon several factors, including the host, the nature and the severity ofthe condition being treated, the mode of administration and theparticular compound employed. However, in general, conditions mediatedby DPP-IV inhibition are effectively treated when a compound of theinvention, or a corresponding pharmaceutically acceptable acid additionsalt, is administered enterally, e.g., orally, or parenterally, e.g.,intravenously, preferably orally, at a daily dosage of 0.002-5,preferably 0.02-2.5 mg/kg body weight or, for most larger primates, adaily dosage of 0.1-250, preferably 1-100 mg. A typical oral dosage unitis 0.01-0.75 mg/kg, one to three times a day. Usually, a small dose isadministered initially and the dosage is gradually increased until theoptimal dosage for the host under treatment is determined. The upperlimit of dosage is that imposed by side effects and can be determined bytrial for the host being treated.

The compounds of the invention, and their corresponding pharmaceuticallyacceptable acid addition salts, may be combined with one or morepharmaceutically acceptable carriers and, optionally, one or more otherconventional pharmaceutical adjuvants and administered enterally, e.g.,orally, in the form of tablets, capsules, caplets, etc. or parenterally,e.g., intravenously, in the form of sterile injectable solutions orsuspensions. The enteral and parenteral compositions may be prepared byconventional means. Examples of suitable formulations comprising thecompounds of the invention are described in the patent applications WO2005/067976 or WO 2006/078593.

Thus the invention also concerns a pharmaceutical composition comprisinga compound according to any of claims 1 to 4 or herein describedcompounds, in free form or in pharmaceutically acceptable acid additionsalt form, together with at least one pharmaceutically acceptablecarrier or diluent.

The pharmaceutical compositions according to the invention are thosesuitable for enteral, such as oral or rectal; transdermal and parenteraladministration to mammals, including man, for the treatment ofconditions mediated by DPP4 activity. Such conditions include impairedglucose tolerance, Type 2 diabetes and obesity.

Thus, the pharmacologically active compounds of the invention may beemployed in the manufacture of pharmaceutical compositions comprising aneffective amount thereof in conjunction or admixture with excipients orcarriers suitable for either enteral or parenteral application.Preferred are tablets and gelatin capsules comprising the activeingredient together with:

a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine;

b) lubricants, e.g., silica, talcum, stearic acid, its magnesium orcalcium salt and/or polyethyleneglycol; for tablets also

c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose and orpolyvinylpyrrolidone; if desired

d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt,or effervescent mixtures; and/or

e) absorbants, colorants, flavors and sweeteners.

Injectable compositions are preferably aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions.

Said compositions may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. Said compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.1-75%, preferably about 1-50%, of the active ingredient.

Suitable formulations for transdermal application include atherapeutically effective amount of a compound of the invention withcarrier. Advantageous carriers include absorbable pharmacologicallyacceptable solvents to assist passage through the skin of the host.Characteristically, transdermal devices are in the form of a bandagecomprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound of the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

Accordingly, the present invention provides pharmaceutical compositionsas described above for the treatment of conditions mediated bydipeptidyl peptidase-IV inhibition, preferably, impaired glucosetolerance, Type 2 diabetes and obesity.

The present invention also provides the use of a pharmaceuticalcompositions as described above for the treatment of conditions mediatedby dipeptidyl peptidase-IV inhibition, preferably, impaired glucosetolerance, Type 2 diabetes and obesity, wherein the compound of theinvention (as defined in any of claims 1 to 4) is administered incombination with another therapeutic agent, e.g. one or two additionalagent, as hereinafter described.

The pharmaceutical compositions may contain a therapeutically effectiveamount of a compound of the invention as defined herein (e.g. in claims1 to 4), either alone or in a combination with another therapeuticagent, e.g., each at an effective therapeutic dose as reported in theart. Such therapeutic agents include:

a) antidiabetic agents, such as insulin, insulin derivatives andmimetics; insulin secretagogues such as the sulfonylureas, e.g.,Glipizide, glyburide and Amaryl; insulinotropic sulfonylurea receptorligands such as meglitinides, e.g., nateglinide and repaglinide; proteintyrosine phosphatase-1B (PTP-1B) inhibitors such as PTP-112; GSK3(glycogen synthase kinase-3) inhibitors such as SB-517955, SB-4195052,SB-216763, N,N-57-05441 and N,N-57-05445; RXR ligands such as GW-0791and AGN-194204; sodium-dependent glucose cotransporter inhibitors suchas T-1095; glycogen phosphorylase A inhibitors such as BAY R3401;biguanides such as metformin; alpha-glucosidase inhibitors such asacarbose; GLP-1 (glucagon like peptide-1), GLP-1 analogs such asExendin-4 and GLP-1 mimetics; and DPPIV (dipeptidyl peptidase IV)inhibitors such as vildagliptin;b) hypolipidemic agents such as 3-hydroxy-3-methyl-glutaryl coenzyme A(HMG-CoA) reductase inhibitors, e.g., lovastatin, pitavastatin,simvastatin, pravastatin, cerivastatin, mevastatin, velostatin,fluvastatin, dalvastatin, atorvastatin, rosuvastatin and rivastatin;squalene synthase inhibitors; FXR (farnesoid X receptor) and LXR (liverX receptor) ligands; cholestyramine; fibrates; nicotinic acid bile acidbinding resins such as cholestyramine; fibrates; nicotinic acid andother GPR109 agonists; cholesterol absorption inhibitors such asezetimibe; CETP inhibitors (cholesterol-ester-transfer-proteininhibitors), and aspirin;c) anti-obesity agents such as orlistat, sibutramine and CannabinoidReceptor 1 (CB1) antagonists e.g. rimonabant; andd) anti-hypertensive agents, e.g., loop diuretics such as ethacrynicacid, furosemide and torsemide; angiotensin converting enzyme (ACE)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, moexipril, perinodopril, quinapril, ramipril andtrandolapril; inhibitors of the Na-K-ATPase membrane pump such asdigoxin; neutralendopeptidase (NEP) inhibitors; ACE/NEP inhibitors suchas omapatrilat, sampatrilat and fasidotril; angiotensin II antagonistssuch as candesartan, eprosartan, irbesartan, losartan, telmisartan andvalsartan, in particular valsartan; renin inhibitors such as ditekiren,zankiren, terlakiren, aliskiren, RO 66-1132 and RO-66-1168; □-adrenergicreceptor blockers such as acebutolol, atenolol, betaxolol, bisoprolol,metoprolol, nadolol, propranolol, sotalol and timolol; inotropic agentssuch as digoxin, dobutamine and milrinone; calcium channel blockers suchas amlodipine, bepridil, diltiazem, felodipine, nicardipine, nimodipine,nifedipine, nisoldipine and verapamil; aldosterone receptor antagonists;and aldosterone synthase inhibitors.e) agonists of peroxisome proliferator-activator receptors, such asfenofibrate, pioglitazone, rosiglitazone, tesaglitazar, BMS-298585,L-796449, the compounds specifically described in the patent applicationWO 2004/103995 i.e. compounds of examples 1 to 35 or compoundsspecifically listed in claim 21, or the compounds specifically describedin the patent application WO 03/043985 i.e. compounds of examples 1 to 7or compounds specifically listed in claim 19 and especially(R)-1-{4-[5-methyl-2-(4-trifluoromethyl-phenyl)-oxazol-4-ylmethoxy]-benzenesulfonyl}-2,3-dihydro-1H-indole-2-carboxylicor a salt thereof.

In each case in particular in the compound claims and the final productsof the working examples, the subject matter of the final products, thepharmaceutical preparations and the claims are hereby incorporated intothe present application by reference to these publications and patentapplications.

Thus the invention covers pharmaceutical compositions comprising;

i) a compound according to any of claims 1 to 4, and

ii) at least one compound selected from

-   -   a) antidiabetic agents,    -   b) hypolipidemic agents,    -   c) anti-obesity agents,    -   d) anti-hypertensive agents,    -   e) agonists of peroxisome proliferator-activator receptors,        ii) one or more pharmaceutically acceptable carriers.

Other specific anti-diabetic compounds are described by Patel Mona inExpert Opin lnvestig Drugs, 2003, 12(4), 623-633, in the FIGS. 1 to 7,which are herein incorporated by reference. A compound of the presentinvention may be administered either simultaneously, before or after theother active ingredient, either separately by the same or differentroute of administration or together in the same pharmaceuticalformulation.

The structure of the therapeutic agents identified by code numbers,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g., PatentsInternational (e.g. IMS World Publications). The corresponding contentthereof is hereby incorporated by reference.

Accordingly, the present invention provides pharmaceutical compositionscomprising a therapeutically effective amount of a compound of theinvention in combination with a therapeutically effective amount ofanother therapeutic agent, preferably selected from anti-diabetics,hypolipidemic agents, anti-obesity agents or anti-hypertensive agents,most preferably from antidiabetics or hypolipidemic agents as describedabove.

The present invention further relates to pharmaceutical compositions asdescribed above for use as a medicament.

The present invention further relates to use of pharmaceuticalcompositions or combinations as described above for the preparation of amedicament for the treatment of conditions mediated by dipeptidylpeptidase-IV inhibition, preferably, impaired glucose tolerance, Type 2diabetes and obesity.

The compounds of the invention, and their corresponding pharmaceuticallyacceptable acid addition salts, may be formulated into enteral andparenteral pharmaceutical compositions containing an amount of theactive substance that is effective for treating conditions mediated byDPP-IV inhibition, such compositions in unit dosage form and suchcompositions comprising a pharmaceutically acceptable carrier.

The compounds of the invention (including those of each of the subscopesthereof and each of the examples) may be administered inenantiomerically pure form (e.g., ee>98%, preferably >99%) or togetherwith the R enantiomer, e.g., in racemic form. The above dosage rangesare based on the compounds of the invention (excluding the amount of theR enantiomer).

The following examples show representative compounds encompassed by thisinvention and their synthesis. However, it should be clearly understoodthat they are for purposes of illustration only.

I. EXAMPLE 1

Pyrrolidine, 1-[(3-hydroxy-1-adamantyl)amino]acetyl-2-cyano-, (S) (INN:vildagliptin) or NVP-LAF237

A. 1-Aminoadamantane-3-ol

Slight modifications to the synthesis found in Khim.-Farm. Zh. (1986),20(7), 810-15, may be used.

To a rapidly stirred, clear and colorless, ice-water chilled mixture ofconcentrated sulfuric acid 96% (210 mL; 3.943 mmol) and 65% nitric acid(21.0 mL; 217.0 mmol) is added 21.0 g (112.0 mmol) of 1-adamantylamineHCl (99%), in small portions over 30 minutes. Upon adamantylaminehydrochloride addition, slight bubbling occurs and the reaction isslightly exothermic. This bubbling, yellow solution is stirred atice-water temperature for about 2 hours and then at room temperature for30 hours. This clear, light yellow reaction is then poured into about100 g of ice and the resulting solution is clear green-blue.

The solution is placed in an ice-water bath and allowed to stir for 30minutes. Approximately 550 g of 89% pure KOH (8.74 mol) is then added insmall portions over 45 minutes. During this addition, the reaction isexothermic; reaching 80° C. and producing copious amounts of brown NO₂gas. By the end of the addition, the reaction is thick with white solids(both product and salts). The resulting white paste is then poured ontoa buchner funnel/celite pad and washed with 1.2 L of CH₂Cl₂. The CH₂Cl₂layer is then extracted from the water layer and dried over Na₂SO₄. Thesolution is then filtered and concentrated (rotovap/pump) to provide1-aminoadamantane-3-ol as a white solid.

B. 1-Chloroacetyl-2-cyanopyrrolidine

To a mechanically stirred solution of 20.0 g (180.0 mmol) ofchloroacetylchloride and 97 g (0.70 mmol) of potassium carbonate in 150mL of tetrahydrofuran is added a solution of L-prolinamide 20.0 g (180.0mmol) in 500 mL of tetrahydrofuran in a dropwise fashion over 45minutes. This reaction is then mechanically stirred for an additionaltwo hours at room temperature. The reaction is then filtered to removepotassium salts and the filtrate is dried over Na₂SO₄. The Na₂SO₄ isthen removed via filtration and to this colorless filtrate is addedtrifluoroacetic anhydride (25.0 mL, 0.180 mmol) in one portion. Thereaction is then magnetically stirred for 1 hour at room temperature andthe resulting clear yellow/orange solution is concentrated via rotovap.The excess trifluoroacetic anhydride is removed by adding ethyl acetateto the concentrated oil and reconcentrating via rotovap. This removingoperation is performed three times.

The resulting oil is partitioned between ethyl acetate and water. Theproduct is then extracted into the ethyl acetate and the aqueous layeris then washed twice with ethyl acetate. The combined organic layers arethen washed successively with water and brine dried over magnesiumsulfate, filtered and concentrated to obtain1-chloroacetyl-2-cyanopyrrolidine as a yellow solid.

C. Pyrrolidine, 1-[(3-hydroxy-1-adamantyl)amino]acetyl-2-cyano-, (S)

To a heterogeneous solution of the title A compound(1-aminoadamantane-3-ol (5.80 g, 34.7 mmol) in CH₂Cl₂ (68.0 mL) is added9.6 g (69 mmol) of K₂CO₃. This heterogeneous mixture is then cooled inan ice-water bath and a solution of 3.0 g (17 mmol) of the title Bcompound (1-chloroacetyl-2-cyanopyrrolidine) dissolved in 25.0 mL ofCH₂Cl₂ is added dropwise over a period of 30 minutes. The resultingmixture is stirred for 2 hours at 0° C. and at room temperature for 6days. The reaction is then concentrated to obtain a yellow pastymaterial which is purified on silica gel employing a SIMS/Biotage Flashchromatography system and a 7% solution of methanol in methylenechloride as the eluent to yield the title compound in free base form asa white crystalline solid (melting point 138° C.-140° C., ¹³CNMR(ppm)=119.59).

A. D. Biocatalytic Synthesis of NVP-BQS867 (the O-glucuronide ofVildaoliptin) and NVP-BRU563 (the Labeled O-Glucuronide ofVildaqliptin).

The reaction scheme of the enzymatic glucuronidation of NVP-LAF237 undercatalysis of rat liver homogenate is shown in FIG. 4-1.

3. 1. Preparation of Rat Liver Homogenate

Two portions of 15 g and one of 11 g of frozen rat liver were defrostedand cut into small pieces. After addition of 0.5 volume equivalent ofice cold 0.9% NaCl solution to each portion of liver and mixing in aDispomix blender, the tissue was homogenized in a “Potter S” TissueHomogenizer (Braun Biotech Inc., Melsungen, Germany) under cooling inice water by moving the teflon pistil 3 times up and down at 100%stirrer speed. The homogenate was filled up to a final weight of 81 gand centrifuged at 4-6° C. for 30 min at 10,000 rpm (=17,000×g) in aBeckmann Coulter centrifuge (Fullerton, Calif., USA) type Avanti J-HCequipped with a JA-10 rotor. The supernatant served as the enzymesource.

4. 2. Bioconversion on Preparative Scale

The conditions for glucuronidation of NVP-LAF237 (vildagliptin) on 340mg scale were: NVP-LAF237 5 mM, UDP-glucuronate (Yamasa Co., Tokyo, JP)20 mM, MgCl₂ 20 mM, HEPES, pH 8.5 160 mM, rat liver homogenate 20% v/v,total volume 224 ml, incubation for 5 h at 37° C. and further 14 h at30° C. The reactions were performed in 50 ml Nunc tubes each filled with20 ml of reaction mixture, which were shaken at 170 rpm on amicrobiological lab shaker with 5 cm shaking radius.

The preparative reaction was stopped by addition of 224 ml ofacetonitrile and mixing for 10 min at 20° C. After centrifugation for 15min at 8000 rpm (rotor JA-10), the pellet was suspended in 50 ml of 50%v/v acetonitrile in deionized water and centrifuged again. The combinedsupernatants were concentrated to 50 ml under reduced pressure at 25° C.The turbid concentrate was centrifuged at 5000 rpm, and after filteringthe supernatant through fiberglass it was subjected to preparative HPLC.

For glucuronidation of [¹³C₅ ¹⁵N] labeled NVP-LAF237 (50 mg scale), thebiotransformation conditions were the same as for NVP-LAF237 on 340 mgscale except that the concentration of UDP-glucuronate was 80 mM, of ratliver homogenate 30% v/v, the filling volume of the Nunc tubes was 16.5ml, the total volume 33 ml, the reaction time 4 h and the shaking speed200 rpm.

The reaction was stopped by addition of 16.5 ml of acetonitrile to eachtube, incubation for 15 min on ice and subsequent mixing. Aftercentrifugation for 15 min at 17,000 rpm (rotor JA-10), the sediment wasresuspended in 20 ml of 50% v/v acetonitrile in deionized water andcentrifuged again. The combined supernatants were concentrated to 10 mlunder reduced pressure at 30° C., again diluted to 25 ml final volumeand centrifuged for final clarification. The pellet was resuspended with5 ml of deionized water, and after centrifugation, the latter twosupernatants were combined and subjected to preparative HPLC.

Measurement of Conversion/Analytical HPLC-DAD:

50 μl of a 19 h samples of the 340 mg batch of NVP-LAF237 and 25 μl of a4 h sample of the batch with [¹³C₅ ¹⁵N] labeled NVP-LAF237 were eachmixed with 200 μl of 50% acetonitrile in water and centrifuged in aSigma 4K15C refrigerated centrifuge. The supernatant was analyzed byanalytical RP₁₈—HPLC-DAD (HPLC-DAD 1100 series from AgilentTechnologies, Basel, Switzerland; two connected columns of the typeChromolith Performance RP-18e 100×4.6 mm with a Chromolith GuardCartridge RP-18e 5×4.6 mm (Merck); flow 2 ml/min; mobile phase A: 3 mMaqueous H₃PO₄, mobile phase B: acetonitrile; gradient from 3% to 15% Bin 5 min; DAD detection at 200-400 nm). Conversion was based on theUV-absorption peak areas at 205 nm of the glucuronide and NVP-LAF237.

5. E. Purification of the Metabolites

a) a) NVP-BQS867-NX-2

A part (5 ml or approximately 10%) of bioconverted raw material was usedfor the preparation of batch NVP-BQS867-NX-2. To this volume 35 ml ofaqueous 10 mM NH₄Ac and 8 ml of methanol were added. The pH was adjustedto 7 with diluted acetic acid. This mixture was then used as injectionsolution for preparative liquid chromatography.

The preparative liquid chromatography-mass spectrometry system consistedof a Waters Autopurification system (Waters Corp. Milford, Mass., USA)with 2525 pump, 2767 sample manager, 2996 photodiode detector, 515make-up pump, ZQ2000 mass spectrometer and MassLynx 4.0 and FractionLynx4.0 software.

An Atlantis dC18, 5 μm, 19 mm×100 mm column (Waters) was employed; flowrate 20 ml/min; mobile phase A: aqueous 10 mM NH₄Ac, pH 7.0; mobilephase B: CH₃CN/CH₃OH 4:1, 10 mM NH₄Ac; gradient program: 0 min. 5% B; 2min. 5% B; 7 min. 20% B; 7.1-12 min. 95% B; 12.1-14 min 5% B; injectionvolume 1-5 ml. The column effluent was split, a very small part wasmixed in-line with make-up liquid 2-propanol/H₂O/HCOOH 400:100:1 andintroduced into the ion source of the mass spectrometer; the restentered the DAD detector recording from 200-600 nm, and subsequently thesample manager for fraction collection.

The mass spectrometer was equipped with ESCi interface used in thepositive ESI mode. A capillary voltage of 3 kV and a cone voltage of 30V was applied; mass range 250-550 Da.

The target fraction was collected time-based from 5.3-6.7 min. Targetfractions from 12 HPLC runs were combined, the organic solvent wasremoved under reduced pressure at 30° C., and the remaining solutionfreeze-dried at −80° C. and 0.2 mbar yielding 15 mg as almost colorlesslyophilizate.

The purity was determined by HPLC-DAD and HPLC-MS (see section 2.2.2):Apart from salts (ammonium acetate) and water the batch NVP-BQS867-NX-2had a purity of >97%.

b) b) NVP-BRU563-NX-1 (Stable Labeled Glucuronide)

An equal volume of 20 mM NH₄Ac solution was added to the aqueous extractto form a NH₄Ac concentration of 10 mM. This mixture was adjusted to pH7.0 with diluted acetic acid and then used as injection solution forpreparative liquid chromatography. The preparative liquidchromatography-mass spectrometry system was described in 2.1.3.1.

An Atlantis Prep dC18 OBD, 5 μm, 30 mm×150 mm column (Waters) wasemployed; flow rate 40 ml/min; mobile phase A: aqueous 10 mM NH₄Ac;mobile phase B: CH₃CN/CH₃OH 4:1+10 mM NH₄Ac, pH 7.0; gradient program: 0min. 5% B; 2 min. 5% B; 7 min. 20% B; 7.1-12 min. 95% B; 12.1-14 min 5%B; injection volume 2-6 ml. The column effluent was split in the sameway as described in 2.1.3.1.

The mass spectrometer was equipped with ESCi interface used in thepositive ESI mode. A capillary voltage of 3 kV and a cone voltage of 30V was applied; mass range 100-1000 Da.

The target fraction was collected time-based from 7.0-9.0 min. Targetfractions from 12 HPLC runs were combined, the organic solvent wasremoved under reduced pressure at 30° C., and the remaining solutionfreeze-dried at −80° C. and 0.2 mbar yielding 63 mg as almost colorlesslyophilizate.

The purity was determined by HPLC-DAD and HPLC-MS (see section 2.2.2):Apart from salts (ammonium acetate) and water the batch NVP-BRU563-NX-1had a purity of >98%.

B. F. Analytics for Structure Identification

1. NMR Spectroscopy

The compounds were dissolved in 5 μl DMSO-d₆ and filled in a 1 mmdiameter NMR tube. From NVP-BQS867, a 1D-¹H spectrum and 2D homo- andheteronuclear spectra (COSY, HSQC, HMBC, ROESY) were obtained. FromNVP-BRU563, a ¹H spectrum was measured. All spectra were recorded at300° K on a Bruker DRX600 spectrometer, using a ¹H {¹³C, ¹⁵N}Microliterprobe. From NVP-BQS867, a ¹³C spectrum was measured on aBRUKER DRX600 spectrometer using a 5 mm ¹³C {¹H} Cryoprobe.

2. 2. HPLC-Mass Spectrometry

The liquid chromatograph consisted of a Waters HPLC Acquity (Waters)equipped with a Waters Acquity 2996 PDA detector. Column: Acquity HPLCBEH C18; 1.7 μm; 1.0×150 mm (Waters); flow rate 0.1 ml/min.; eluent A:H₂O/TFA 100:0.1; eluent B: acetonitrile/TFA 100:0.1; gradient: 0 min. 5%B; 1 min. 5% B, 11 min. 40% B; 13-16 min. 95% B; 30° C.; UV-detection:200-350 nm, resolution 2.4 nm; injection volume 5 μl. The substance wasdissolved in water/acetonitrile 9:1 at a concentration of approximately0.3 mg/ml. Evaluation of the purity was done by UV at 210 nm as area-%of the desired peak. The column effluent was introduced directly intothe ion source of the MS.

A TSQ Quantum AM mass spectrometer (Thermo, San Jose, Calif., USA)equipped with electrospray interface in the positive mode was used. Itwas operated with Xcalibur software version 2.0. A sheath gas setting of25 units and auxiliary gas of 5 units was used and a spray voltage of 3kV applied. The heated metal capillary was maintained at 300° C.; massrange 200 to 800 Da. MS/MS parameters: collision gas 1.5 mTorr argon;collision energy 25 V.

II. G. RESULTS

A. 1. Biocatalytic Synthesis

For the two preparative reactions analytical HPLC-DAD gave the followingconversion values: 66% for the batch with 340 mg of NVP-LAF237 and 94%for the batch with [¹³C₅ ¹⁵N] labeled NVP-LAF237 (50 mg).

2. Purification of the Glucuronides

As described in above section, 15 mg of O-glucuronide NVP-BQS867-NX-2was obtained by preparative HPLC-MS from 5 ml of aqueous culture extractobtained from biotransformation. From the whole preparative reactionwith 340 mg of NVP-LAF237, four batches of NVP-BQS867-NX (NX-1 to NX-4)were isolated with a total amount of 295 mg of O-glucuronide.

3. Structure Elucidation

The mass spectrum of NVP-BQS867-NX-2 shows a [M+H]⁺ of 480.1 suggestinga molecular weight of 479. This is consistent with a glucuronide ofNVP-LAF237. The MS/MS product ion spectrum shows the loss of glucuronide(m/z 304) as well as glucuronic acid (m/z 286).

The structure of NVP-BQS867-NX-2 (FIG. 3-1) was elucidated unambiguouslyon the basis of NMR spectroscopy. The major differences in the NMRspectra of NVP-BQS867 compared with NVP-LAF237 are mainly the resonancesobtained from the glucuronide moiety. The ¹H-(4.42 ppm) and the ¹³Cshift (96.4 ppm) of the anomeric 1′ resonance of the glucuronic acidindicated that the latter is attached to oxygen and not to nitrogen. AnHMBC correlation from H-1′ to C-3 and ROESY correlations from H-1′ toH-2 and H-4 confirmed the structure shown in FIG. 3-1 unambiguously.

The interpretation of all NMR data and correlations lead to only onestructure which is in accordance with the MS result (MW=479, m/zMH⁺=480.1). A summary of all ¹H— and ¹³C shifts and relevant homo- andheteronuclear correlations is shown in Table 3-1.

The compound NVP-BRU563-NX-3, biosynthesized from[U-pyrrolidin,cyano-¹³C₅,pyrrolidin-¹⁵N] labeled NVP-LAF237, showedidentical spectral properties to NVP-BQS867-NX with the expectedexceptions for the stable label: The MH⁺ of 486.2 is 6 Da higher thanthat of NVP-BQS867-NX and in the MS/MS product ion spectrum somefragments are also shifted.

-   -   (i) Figure II-1 Structure of NVP-BQS867 with numbering scheme        used in Table 3-1 and ¹H spectrum

Table II-1 NVP-BQS867

(b) ¹H- and ¹³C assignments for structure of NVP-BQS867 HMBC NOE Pos.Group ¹H-shift ¹³C-shift correlation correlations  1 Cq  53.6 H-10, H-2,H-5  2 CH₂ 1.74, 1.60  46.6 H-1′  3 Cq  75.4 H-1′, H-2, H-4  4 2 × 1.70,1.67, 41.9, 41.6 H-1′ CH₂ 1.60  5 2 × 1.55 41.4, 41.1 CH₂  6 2 × CH 2.2030.5, 30.6  7 CH₂ 1.48, 1.43  35.5 10 CH₂ 3.53, 3.49  43.5 11 CO 170.9H-10 13 CH₂ 3.64, 3.46  45.7 14 CH₂ 2.06, 1.99  25.2 15 CH₂ 2.20, 2.13 30.0 16 CH 4.76  47.2 17 Cq 119.9 H-16, H-15  1′ CH 4.42  96.4 H-2′,H-5′ H-2, H-4, H-2′, H-3′, H-5′  2′ CH 2.89  73.8  3′ CH 3.15  77.4  4′CH 3.18  72.7  5′ CH 3.41  74.4  6′ CO 172.6

Another preferred compound is:

In a most preferred embodiment, the compound of Figure BB is in asubstantially pure form.

This O-glucuronide compound (Figure BB) can be obtained by adapting theherein described process. The process to obtain the starting compoundfrom wherein R′ is —OH is described in the patent application WO01/068603, or WO 05/095339.

A salt can be the HCL salt. (HCl)=as hydrochloride. All HCl salts offinal products are prepared by passing HCl gas through a 0.1 Molarsolution of the free base in tetrahydrofuran until solution is clearlyacidic followed by removal of the solvent (rotovap/pump).

The amino-adamantane starting materials are known in the literature orcan be prepared as follows:

The manufacture of 3,5-dimethyl-1-adamantylamine is described in J. Med.Chem., 25; 1; 1982; 51-56.

The manufacture of 3-ethyl-1-adamantylamine is described in J. Med.Chem., 25; 1; 1982; 51-56.

3-Methoxy-1-adamantylamine can be prepared as follows:

To a stirred, ice-water chilled suspension of potassium hydride (0.680gm; 5.95 mmol) in 15.0 ml of tetrahydrofuran is added a mixture of1-aminoadamantane-3-ol (1.00 g; 5.95 mmol) and 15.0 ml oftetrahydrofuran dropwise over 30 minutes. The resulting mixture is thenstirred for an addition 30 minutes and iodomethane (0.370 ml; 5.95 mmol)is then added dropwise over one minute. The resulting opaque whitereaction is then stirred at room temperature for 18 hours. The mixtureis then diluted with 50 ml of methylene chloride and filtered to removethe inorganic impurities. The filtrate is then concentrated and purifiedon silica gel employing a SIMS/Biotage apparatus and 19% methanol and 1%ammonium hydroxide in methylene chloride as eluent to yield3-methoxy-1-adamantylamine as an opaque oil.

Synthesis of 3-[[(tertbutylamino)carbonyl]oxy]-1-aminoadamantane

To a mixture of 1-aminoadamantane-3-ol (5.00 g; 30.0 mmol) and potassiumcarbonate (6.20 g; 45 mmol) in 150 ml of tetrahydrofuran is addedbenzylchloroformate (4.70 g, 33.0 mmol) in dropwise fashion over a 10minute period. The mixture is then stirred at room temperature for 2 hand then partitioned between ethyl acetate and water. The product isthen extracted into the ethyl acetate and the aqueous layer is washedtwice with ethyl acetate (100 ml). The combined organic layers are thenwashed successively with 100 ml of aqueous 2 N sodium hydroxide, waterand brine, dried over sodium sulfate, filtered and concentrated(rotovap/pump) to provide 1-benzylcarbamoyladamantane-3-ol as a whitesolid in 85% yield.

To a clear solution of 1-benzylcarbamoyladamantane-3-ol (1.00 g: 3.32mmol) and tert-butylisocyanate (380 μl, 3.32 mmol) in 30 ml of methylenechloride is syringe-added trimethylsilyl chloride (20.0 μl, 0.17 mmol).This reaction is then stirred at room temperature for 18 hours,concentrated (rotovap) and purified on silica gel employing aSIMS/Biotage apparatus and 20% ethyl acetate in hexane as eluent toyield 3-[[(tertbutylamino)carbonyl]oxy]-1-benzylcarbamoyladamantane as awhite solid in quantitative yield.

To a mixture of3-[[(tertbutylamino)carbonyl]oxy]-1-benzylcarbamoyladamantane (1.50 g,3.75 mmol) and 10% palladium on carbon (400 mg) in ethanol (150 ml) in a1-liter parr hydrogenation flask is added hydrogen (50 psi). This opaqueblack mixture is then shaken for 24 h. The reaction is then filteredthrough celite to remove the palladium catalyst and concentrated(rotovap/pump) to provide3-[[(tertbutylamino)carbonyl]oxy]-1-aminoadamantane as a clear oil in99% yield. The procedure for the synthesis of4-[[[(methoxyphenyl)amino]carbonyl]oxy]-1-aminoadamantane is essentiallythe procedure of 3-[[(tertbutylamino)carbonyl]oxy]-1-aminoadamantaneexcept in the second step where an equivalent of 4-methoxyphenylisocyanate replaces tert-butylisocyanate, 1,2-dichloroethane is used assolvent instead of methylene chloride and the reaction is stirred at 50°C. for 18 hours. The final amine intermediate is provided as an oil.

The procedure for the synthesis of3-[[(phenylamino)carbonyl]oxy]-1-aminoadamantane is essentially theprocedure of 3-[[(tertbutylamino)carbonyl]oxy]-1-aminoadamantane exceptin the second step where an equivalent of phenyl isocyanate replaces thetert-butylisocyanate, 1,2-dichloroethane is used as solvent instead ofmethylene chloride and the reaction is stirred at 50° C. for 18 hours.The final amine intermediate is provided as a clear oil.

The procedure to make 2-aminoadamantane-5-ol is the same as in Example 1except that the starting material is 2-aminoadamantane instead of1-aminoadamantane.

The procedure for the synthesis of the nucleophile3-acetoxy-1-aminoadamantane is essentially the procedure of3-[[(tertbutylamino)carbonyl]oxy]-1-aminoadamantane except for astandard acylation of 1-benzylcarbamoyladamantane-3-ol using 1.2 eq ofacetyl chloride, 3.0 eq. of pyridine, 0.1 eq of 4-dimethylaminopyridineand 1,2 dichloroethane which are all stirred at room temperature for 24hours. The final amine is provided as a thick oil.

The procedure for the synthesis of3-[[[(diisopropyl)amino]carbonyl]oxy]-1-amino-adamantane is essentiallythe procedure of 3-[[(tertbutylamino)carbonyl]oxy]-1-aminoadamantaneexcept in the second step where an equivalent of diisopropylcarbamoylchloride replaces the tert-butylisocyanate, 1,2-dichloroethane is usedas solvent instead of methylene chloride and the reaction is stirred at85° C. for 18 hours. The final amine intermediate is provided as a graysolid.

The procedure for the synthesis of3-[[[(cyclohexyl)amino]carbonyl]oxy]-1-aminoadamantane is essentiallythe procedure of 3-[[(tertbutylamino)carbonyl]oxy]-1-aminoadamantaneexcept in the second step where an equivalent of cyclohexylisocyanatereplaces the tert-butylisocyanate, 1,2-dichloroethane is used as solventinstead of methylene chloride and the reaction is stirred at 50° C. for18 hours.

The final amine intermediate is provided as a thick clear oil.

The procedure to make 3-ethoxy-1-adamantylamine (a clear oil) is thesame as for 3-methoxy-1-adamantylamine except that iodoethane (1.3equivalent) is used instead of iodomethane.

FORMULATION EXAMPLE

Tablets, each containing 50 mg of the active ingredient, NVP-BQS867, canbe prepared as follows:

Composition (for 10,000 tablets)

Active ingredient 500.0 g Lactose 500.0 g Potato starch 352.0 g GelatinTalc 60.0 g Magnesium stearate 10.0 g Silica (highly disperse) 20.0 gEthanol q.s.

The active ingredient is mixed with the lactose and 292 g of potatostarch, and the mixture is moistened using an alcoholic solution of thegelatin and granulated by means of a sieve. After drying, the remainderof the potato starch, the talc, the magnesium stearate and the highlydisperse silica are admixed and the mixture is compressed to givetablets of weight 145.0 mg each and active ingredient content 50.0 mgwhich, if desired, can be provided with breaking notches for fineradjustment of the dose.

III. EXAMPLE 2 Biological Experimental

List of abbreviations Abbreviation Description AMC aminomethylcoumarinBSA bovine serum albumin CHAPS 3-((3-cholamidopropyl)-dimethylamino)-propanesulfonate DABCYL 4-((4-(dimethylamino)phenyl)azo)benzoic acidDMSO dimethylsulfoxide DPP-2 dipeptidyl peptidase 2 DPP-8 dipeptidylpeptidase 8 DPP-9 dipeptidyl peptidase 9 DPP-IV dipeptidyl peptidase IVEDANS 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid FAP fibroblastactivation protein, alpha FI fluorescence intensity HCl hydrochloricacid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid M_(W) eff.efficient molecular weight Nle norleucine NVP-BQS867 NVP-BQS867-NX-2 PEPprolyl-endopeptidase prep. HPLC preparative High-performance liquidchromatography Rh110 rhodamine-110 SD standard deviation Tristris-(hydroxymethyl)-aminomethane

IV. MATERIAL AND METHODS

A. A. Instrumentation

All protein and peptide containing solutions were handled in siliconizedtubes (Life Systems Design, Merenschwand, Switzerland). The compoundsolutions as well as the enzyme and the substrate solutions weretransferred to 384-well plates (black Cliniplate; cat. no. 95040020Labsystems Oy, Finland) by means of a CyBi-Well 96-channel pipettor(CyBio AG, Jena, Germany).

1. 1. Instrumentation for FI Measurements

For fluorescence intensity (FI) measurements with AMC as dye, an UltraEvolution reader (TECAN, Maennedorf, Switzerland) was used. Theinstrument was equipped with a combination of a 350 nm (20 nm bandwidth)and a 500 nm (25 nm bandwidth) bandpath filter for fluorescenceexcitation and emission acquisition, respectively. To increase thesignal:background ratio, an appropriate dichroic mirror was employed.All filters and the dichroic mirror were purchased from TECAN.

FI measurements using a Rh110 dye were done with a Safire2 reader(TECAN, Maennedorf, Switzerland). The Safire2 is a monochomator-basedinstrument and wavelengths of 485 nm and 535 nm were taken forfluorescence excitation and emission acquisition, respectively. Thebandwidths were set to 20 nm in both the excitation and the emissionpath.

The fluorophores in each well were excited by three flashes permeasurement.

2. Calculation of IC₅₀ Values from Averaged Data

The data from the independent assay runs was averaged and plotted usingprogram of Origin 7.5SR6 (OriginLab Corporation, Northampton, Mass.,USA). Origin's built-in non-linear regression routine was used to fitthe averaged data to the ‘logistics’ functiony=A2+(A1−A2)/(1+(x/IC ₅₀)^p)  (Equation 1)where y is the %-inhibition at the inhibitor concentration, x. A1 is thelowest inhibition value, i.e. 0%, and A2 the maximum inhibition value,i.e. 100%. The exponent, p, is the Hill coefficient.B. Determination of IC₅₀ Values

For the determination of IC₅₀ values, the assays were performed at roomtemperature in 384-well plates. All final assay volumes were 30 □l. Testcompounds were dissolved in 90% (v/v) DMSO/water and diluted in water(containing 0.05% (w/v) CHAPS) to 3-times the desired assayconcentration. For each assay, 10 □l water/CHAPS (±test compound) wereadded per well, followed by 10 □l protease solution (diluted with assaybuffer; for final assay concentration cf. § Assay conditions). After 1hour of incubation at room temperature, the reaction was started byaddition of 10 □l substrate solution (for final concentrations cf. §Assay conditions). The eleven final compound concentrations were either0.9 nM, 3 nM, 9 nM, 30 nM, 90 nM, 300 nM, 900 nM, 3 μM, 9 μM, 30 μM and90 μM or 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, 30 μM,100 μM and 300 μM.

The effect of the compound on the enzymatic activity was obtained fromthe linear progress curves and determined from two readings, the firstone taken directly after the addition of substrate (t=0 min) and thesecond one after 1 hour (t=60 min). The IC₅₀ value was calculated fromthe plot of percentage of inhibition vs. inhibitor concentration usingnon-linear regression analysis software (XLfit, Vers. 4.0; ID BusinessSolution Ltd., Guildford, Surrey, UK).

C. Assay Conditions

All conditions in respect to enzyme, substrate and buffer are listedbelow for the individual assays.

1.—DPP-2 (Dipeptidyl Peptidase 2)

enzyme: human DPP-2 covering amino acid 30-492; expressed in andpurified from insect cells (baculovirus expression system)

substrate: Nle-Pro-AMC, purchased from Biosyntan (www.biosyntan.de),product number 4572

enzyme concentration: 0.03 nM

substrate concentration: 2 □M

assay buffer: 100 mM sodium citrate, pH 5.5, 0.05% (w/v) CHAPS

2.—DPP-IV (Dipeptidyl Peptidase IV)

enzyme: human DPP-IV covering amino acid 39-766; expressed in andpurified from insect cells (baculovirus expression system)

substrate: Gly-Pro-AMC, purchased from Bachem (www.bachem.com),catalogue number 1-1225

enzyme concentration: 0.01 nM

substrate concentration: 10 □M

assay buffer: 25 mM Tris, pH 7.4, 140 mM NaCl, 10 mM KCl, 0.05% (w/v)CHAPS

3.—Human Plasma DPP-IV (Endogenous Dipeptidyl Peptidase IV in HumanBlood Plasma)

enzyme: endogenous human DPP-IV from plasma sample, male donors

substrate: (H-Ala-Pro)₂—Rh110, in house synthesis

enzyme concentration: approx. 5 nM

substrate concentration: 10 □M

assay solution: human blood plasma, diluted to 50% plasma content withbuffer (25 mM Tris, pH 7.4, 140 mM NaCl, 10 mM KCl, 0.05% (w/v) CHAPS)

4.—DPP-8 (Dipeptidyl-Peptidase 8)

enzyme: human DPP-8 covering amino acid 1-882; expressed in and purifiedfrom insect cells (baculovirus expression system)

substrate: Gly-Pro-AMC, purchased from Bachem (www.bachem.com),catalogue number 1-1225

enzyme concentration: 0.05 nM

substrate concentration: 10 □M

assay buffer: 25 mM Tris, pH 7.4, 140 mM NaCl, 10 mM KCl, 0.05% (w/v)CHAPS

5.—DPP-9 (Dipeptidyl-Peptidase 9)

enzyme: human DPP-9 covering amino acid 1-863; expressed in and purifiedfrom Pichia pastoris

substrate: Gly-Pro-AMC, purchased from Bachem (www.bachem.com),catalogue number 1-1225

enzyme concentration: 1 nM

substrate concentration: 10 □M

assay buffer: 25 mM Tris, pH 7.4, 140 mM NaCl, 10 mM KCl, 0.05% (w/v)CHAPS

6.—FAP (Fibroblast Activation Protein, Alpha)

enzyme: human FAPα covering amino acid 27-760 excluding the cytoplasmicand transmebrane domains; expressed in and purified from insect cells(baculovirus expression system)

substrate: Z-Gly-Pro-AMC, purchased from Bachem (www.bachem.com),catalogue number 1-1145

enzyme concentration: 0.1 nM

substrate concentration: 8 □M

assay buffer: 100 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.05% (w/v)CHAPS

7.—PEP (Prolyl-Endopeptidase)

enzyme: human PEP covering amino acid 1-710; expressed in and purifiedfrom Pichia pastoris

substrate: Z-Gly-Pro-AMC, purchased from Bachem (www.bachem.com),catalogue number 1-1145

enzyme concentrations: 0.03 nM

substrate concentration: 100 □M

assay buffer: 100 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.05% (w/v)CHAPS, 0.1% (w/v) BSA.

V. D. RESULTS

The results of all IC₅₀ measurements for the compound NVP-BQS867 aresummarized in the Tables 3-1 to 3-7 below. For hDPP-IV, endogenousDPP-IV in human blood plasma, hDPP-8, hDPP-9, hFAP and hPEP.

TABLE V-1 (a) Potency of NVP-BQS867-NX-2 on human DPP-2. Mean IC₅₀ value± SD Experiment IC₅₀ value [□M] Hill coefficient [□M] 1 >300 — >902 >300 — 3 >90 — 4 >90 —

TABLE V-2 (b) Potency of NVP-BQS867-NX-2 on human DPP-IV. Mean IC₅₀value ± SD Experiment IC₅₀ value [□M] Hill coefficient [□M] 1 0.004 0.70.006 ± 0.001 2 0.006 0.9 3 0.007 1.3 4 0.005 1.1

TABLE V-3 (c) Potency of NVP-BQS867-NX-2 on human plasma DPP-IV. MeanIC₅₀ value ± SD Experiment IC₅₀ value [□M] Hill coefficient [□M] 1 0.0031.4 0.004 ± 0.001 2 0.004 1.4 3 0.004 1.2 4 0.004 1.2

TABLE V-4 (d) Potency of NVP-BQS867-NX-2 on human DPP-8. Mean IC₅₀ value± SD Experiment IC₅₀ value [□M] Hill coefficient [□M] 1 8.3 1.2 8.0 ±0.3 2 8.0 1.3 3 8.1 1.2 4 7.6 1.2

TABLE V-5 (e) Potency of NVP-BQS867-NX-2 on human DPP-9. Mean IC₅₀ value± SD Experiment IC₅₀ value [□M] Hill coefficient [□M] 1 0.7 1.0 0.5 ±0.2 2 0.4 0.9 3 0.6 1.0 4 0.4 0.9

TABLE V-6 (f) Potency of NVP-BQS867-NX-2 on human FAP. Mean IC₅₀ value ±SD Experiment IC₅₀ value [□M] Hill coefficient [□M] 1 43 1.3 24 ± 13 216 0.8 3 19 0.7 4 19 0.8

TABLE V-7 (g) Potency of NVP-BQS867-NX-2 on human PEP. Mean IC₅₀ value ±SD Experiment IC₅₀ value [□M] Hill coefficient [□M] 1 52 0.7 25 ± 18 215 1.2 3 18 0.8 4 14 0.6

What is claimed is:
 1. A method of inhibiting dipeptidyl peptidase-IVcomprising administering to a mammal in need of such treatment atherapeutically effective amount of a compound of formulae (I A), (I B),(X A), (X B), (Y A) or (Y B)

wherein R′ represents

and R″ represents hydrogen, hydroxy, C₁-C₇alkoxy, C₁-C₈-alkanoyloxy, orR₅R₄N—CO—O—, where R₄ and R₅ independently are C₁-C₇alkyl or phenylwhich is unsubstituted or substituted by a substitutent selected fromC₁-C₇alkyl, C₁-C₇alkoxy, halogen and trifluoromethyl and where R₄additionally is hydrogen; or R₄ and R₅ together represent C₃-C₆alkylene;in free form of a pharmaceutically acceptable acid addition saltthereof.
 2. A method of inhibiting dipeptidyl peptidase-IV comprisingadministering to a mammal in need of such treatment a therapeuticallyeffective amount of a pharmaceutical composition comprising atherapeutically effective amount of the compound of formula (I A), (IB), (X A), (X B), (Y A) or (Y B) of claim 1 together with at least onepharmaceutically acceptable carrier or diluent.
 3. The method accordingto claim 1 wherein the mammal in need of such treatment hasnon-insulin-dependent diabetes mellitus.
 4. The method according toclaim 2 wherein the mammal in need of such treatment hasnon-insulin-dependent diabetes mellitus.